Endothelial dysfunction from reduced endothelium-derived nitric oxide bioavailability is a hallmark of the abnormal vascular function observed in individuals with hypercholesterolemia and other risk factors for atherosclerosis. Endothelial function is assessed by measuring vasodilation in response to agonists such as bradykinin that promotes release of nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF). The contribution of nitric oxide to human vascular tone and function in vivo has been extensively studied, but investigation of EDHF in vivo has been hampered because of controversy surrounding the true identity of EDHF. Recent experimental studies have demonstrated several potential mechanisms underlying endothelium-dependent hyperpolarization; these include (1) release of epoxides from cytochrome P450 (CYP450)-dependent metabolism of arachidonic acid, (2) promotion of endothelial and smooth muscle hyperpolarization by stimulation of small and large calcium-dependent potassium channels (K+cA) on endothelial cells, (3) release of hydrogen peroxide synthesized by dismutation of superoxide anions, and (4) activation of gap junctions. The aim of this proposal is to establish the activity and identity of EDHF in the human forearm circulation, investigate its physiologic role in vasodilation, and study whether its activity is modulated by hypercholesterolemia. Employing specific inhibitors of CYP450, K+CA channels, and hydrogen peroxide, and by measuring forearm vasodilation at rest and with bradykinin, after inhibition of nitric oxide and cyclooxygenase, we will test the following hypotheses in healthy and hypercholesterolemic subjects: Specific Aim l: EDHF activity is due to CYP 450-derived epoxide release; Specific Aim 2: EDHF mediates vasodilation by activation of K+CA channels; Specific Aim 3: EDHF activity is due to hydrogen peroxide release; and Specific Aim 4: whether physiologic vasodilation during sustained hyperemia or exercise, that is known to have a minimal contribution from nitric oxide and prostacyclin, is due to EDHF release.